Abstract [en]

Within the field of depression the “monoamine hypothesis” has been the leading theory to explain the biological basis of depression. This theory proposes that the biological basis of depression is due to a deficiency in one or more of three key neurotransmitter systems, namely noradrenaline, dopamine and serotonin which are thought to mediate the therapeutic actions of virtually every known antidepressant agent.

Citalopram is a selective serotonin-reuptake inhibitor (SSRI) used for the treatment of depression and anxiety disorders. Citalopram is a racemic compound, in other words composed of a 50:50 mixture of two enantiomers (S-(+)-citalopram and R-(-)-citalopram) and with one of the enantiomers (S-(+)-citalopram) accounting for the inhibitory effect. At the time of introduction of citalopram the physician needed a therapeutic drug monitoring service to identify patients with interactions, compliance problems and for handling questions concerning polymorphic enzymes and drug metabolism. An achiral analytical separation method based on solid-phase extraction followed by high-performance liquid chromatography (HPLC) was developed for routine therapeutic drug monitoring (TDM) of citalopram and its two main demethylated metabolites.

As the data available on citalopram were from achiral concentration determinations and to be able to further investigate citalopram enantiomers effects and distribution, a chiral method for separation of the enantiomers of citalopram and its demethylated metabolites was established. The advances within chiral separation techniques have made measurement of the concentrations of the individual enantiomers in biological fluids possible.

The process behind enantioselective separation is however not fully understood and the mechanism behind the separation can be further scrutinized by the use of multivariate methods. A study of the optimization and characterization of the separation of the enantiomers of citalopram, desmethylcitalopram and didesmethylcitalopram on an acetylated ß-cyclodextrin column, by use of two different chemometric programs - response surface modelling and sequential optimization was performed. Sequential optimization can be a quicker mean of optimizing a chromatographic separation; response surface modelling, in addition to enabling optimization of the chromatographic process, also serves as a tool for learning more about the separation mechanism.

Studies of the antidepressant effect and pharmacokinetics of citalopram have been performed in adults, but the effects on children and adolescents have only been studied to a minor extent, despite the increasing use of citalopram in these age groups.

A study was initiated to investigate adolescents treated for depression, with respect to the steady-state plasma concentrations of the enantiomers of citalopram and its demethylated metabolites. The ratios between the S- and R-enantiomers of citalopram and didesmethylcitalopram were in agreement with studies involving older patients. The concentrations of the S-(+)- and R-(-) enantiomers of citalopram and desmethylcitalopram were also in agreement with values from earlier studies. The results indicate that the use of oral contraceptives may have some influence on the metabolism of citalopram. This might be because of an interaction of the contraceptive hormones with the polymorphic CYP2C19 enzyme.

Even though the SSRIs are considered less toxic compared with older monoamine-active drugs like the tricyclic/tetracyclic antidepressants, the risk of developing serious side effects such as ECG abnormalities and convulsions has been seen for citalopram, when larger doses have been ingested. Furthermore, fatal overdoses have been reported where citalopram alone was the cause of death. Data on the toxicity of each of the enantiomers in humans have not been reported and no data on blood levels of the enantiomers in cases of intoxication have been presented.

An investigation was initiated on forensic autopsy cases where citalopram had been found at the routine screening and these cases were further analysed with enantioselective analysis to determine the blood concentrations of the enantiomers of citalopram and metabolites. Furthermore the genotyping regarding the polymorphic enzymes CYP2D6 and CYP2C19 were performed.

In 53 autopsy cases, we found increasing S/R ratios with increasing concentrations of citalopram. We found also that high citalopram S/R ratio were associated with high parent drug to metabolite ratio and may be an indicator of recent intake. Only 3.8 % were found to be poor metabolizers regarding CYP2D6 and for CYP2C19 no poor metabolizer was found.

Enantioselective analysis of citalopram and its metabolites can provide valuable information about the time that has elapsed between intake and death. Genotyping can be of help in specific cases but the possibility of pharmacokinetic interactions is apparently a far greater problem than genetic enzyme deficiency.

Abstract [en]

An assay based on solid-phase extraction followed by high-performance liquid chromatography (HPLC) was developed for the measurement of citalopram and its main metabolites desmethylcitalopram and didesmethylcitalopram. The best extraction procedure was performed with end-capped C2 column utilising secondary silanol interactions to obtain clean extract. The HPLC analysis was done on a phenyl column with a mobile phase without any amine additives. Fluorescence detection gave a limit of detection of 0.8 nmol/l plasma for the compounds analysed.

Abstract [en]

Response-surface modelling and sequential optimization have been used for optimization and characterization of the separation of the enantiomers of citalopram, desmethylcitalopram, and didesmethylcitalopram on an acetylated ▀-cyclodextrin column. In the model chosen the separation conditions mobile phase methanol content, buffer concentration, column temperature, and pH were varied to investigate their influence on the chromatography. It was found that what is good for selectivity within an enantiomer pair is bad for selectivity between enantiomer pairs. Because within-pair and between-pair selectivity does not reach its optimum at the same conditions, a middle course approach has to be followed. Use of an experimental design for this investigation enabled understanding of the mechanisms of within- and between-pair separation for citalopram, desmethylcitalopram, and didesmethylcitalopram. Sequential optimization can be a quicker means of optimizing a chromatographic separation, response-surface modelling, in addition to enabling optimization of the chromatographic process, also serves as a tool for learning more about the separation mechanism.

Abstract [en]

Studies of the antidepressant effect and pharmacokinetics of citalopram have been performed in adults, but the effects on children and adolescents have only been studied to a minor extent despite its increasing use in these age groups. The aim of this study was to investigate a group of adolescents treated for depression, with respect to the steady-state plasma concentrations of the enantiomers of citalopram and its demethylated metabolites desmethylcitalopram and didesmethylcitalopram. Moreover, the authors studied the genotypes for the polymorphic cytochrome P450 enzymes CYP2D6 and CYP2C19 in relation to the different enantiomers. The S/R ratios of citalopram and desmethylcitalopram found in this study of 19 adolescents were similar to studies involving older patients. The concentrations of the R-(-)- and S-(+)-enantiomers of citalopram and desmethylcitalopram were also in agreement with values from earlier studies, the R-(-)-enantiomer (distomer) being the major enantiomer. The results indicate that the use of oral contraceptives may have some influence on the metabolism of citalopram. This might be because of an interaction of the contraceptive hormones with the CYP2C19 enzyme.

Abstract [en]

Citalopram, a selective serotonin reuptake inhibitor, is one of the most commonly found drugs in Swedish forensic autopsy cases. Citalopram is a racemic drug with 50:50 of the S- and R- enantiomers. Enantioselective analysis of citalopram and its metabolites desmethylcitalopram and didesmethylcitalopram were performed in femoral blood from 53 autopsy cases by a chiral high-performance liquid chromatography (HPLC) method. The mean (± standard deviation) S/R ratio for citalopram was 0.67 ± 0.25 and for desmethylcitalopram, 0.68 ± 0.20. We found increasing S/R ratios with increasing concentrations of citalopram. We also found that high citalopram S/R ratios were associated with a high parent drug-to-metabolite ratio and may be an indicator of recent intake. Citalopram is metabolized by cytochrome P450 (CYP) 3A4, 2C19, and 2D6. Genotyping for the polymorphic CYP2C19 and CYP2D6 revealed no poor metabolizers regarding CYP2C19 and only 2 (3.8%) poor metabolizers regarding CYP2D6. The presence of drugs metabolized by and/or inhibiting these enzymes in several of the cases suggests that such pharmacokinetic interactions are a more important (practical) problem than metabolic deficiency. Enantioselective analysis of citalopram and its metabolites can provide additional information when interpreting forensic toxicology results and might be a necessity in the future.